Modified Acrylic Block Copolymers For Hydrogels and Pressure Sensitive Wet Adhesives

ABSTRACT

A method of producing an acrylic block copolymer comprising hydrophobic poly (lower alkyl methoacrylate), hydrophilic poly (lower alkyl methacrylic acid), and hydrophobic poly (lower alkyl methacrylate).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. patent applicationSer. No. 11/676/099, filed Feb. 16, 2007, entitled “Modified AcrylicBlock Copolymers for Hydrogels and Pressure Sensitive Wet Adhesives”,which claims the priority date of provisional patent application60/773,910, filed Feb. 16, 2006, is claimed herein. The disclosure ofthe 60/773,910 application is also incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made, at least in part, with United States Governmentsupport awarded by the following agencies: National Institutes of Health(NIH) Grant No. R01 DE14193, Cufs No. 0600-350-K301; National ScienceFoundation (NSF) Grant No. 0214146, Cufs No. 0830-250-w646. The UnitedStates Government may have certain rights in this invention. And HumanFrontier Science Program, Cufs No. 0995-350-w233.

BACKGROUND OF THE INVENTION

There is a growing demand for bioadhesives that can be easily deliveredand that solidify in situ to form strong and durable interfacialadhesive bonds and are resistant to the normally detrimental effects ofwater. Some of the potential applications for such biomaterials includeconsumer adhesives, bandage adhesives, tissue adhesives, bonding agentsfor implants, and drug delivery. It is also preferable to prepare theseadhesives in a toxicologically acceptable solvent that enables injectionto the desired site and permits a conformal matching of the desiredgeometry at the application site.

BRIEF SUMMARY OF THE INVENTION

Briefly, in one aspect, the present invention addresses, in part, theabove demand with a modified acrylic block, especially triblock,copolymer system, which can be fully dissolved in toxicologicallyacceptable organic solvents. In this approach, hydrophilic andhydrophobic lower alkyl methacrylate copolymer “blocks” are chosen orare created so that hydrogels can be formed by a solvent exchangemechanism when a solution of the block copolymer in an acceptablesolvent is exposed to water that is naturally present within the bodyand gels. By this process in situ formation of a bioadhesive in anaqueous environment is accomplished. “Lower alkyl” will be understood byone skilled in this art generally to mean having about 1 to 6 carbonatoms and being predominantly but not necessarily exclusivelyhydrocarbon in nature. Preferred lower alkyl moieties herein are methyland tert-butyl.

In one embodiment of this invention poly(methyl methacrylate—tert-butylmethacrylate- methyl methacrylate) (PMMA-PtBMA-PMMA) triblock copolymeris synthesized by anionic polymerization. The PtBMA midblock is thenconverted to hydrophilic poly-methacrylic acid (PMAA).

In a further embodiment of this invention, the above block (co)-polymerswere modified with L-3,4-dihydroxyphenylalanine (DOPA), a modified aminoacid that is believed to be responsible for wet adhesion in musseladhesive proteins. The preferred triblock polymer, so modified, wasfully dissolved in N-methyl pyrrolidone (NMP), dimethyl sulfoxide(DMSO), or dimethylformamide (DMF), and hydrogels were formed byexposing the solutions to saturated water vapor.

It is significant to note that the preferred PtBMA, after conversion topMAA, noted above has the advantage of being easily modified to haveother functional groups such as —NH₂, —OH. The —COOH and —OH derivativesare particularly preferred because they permit ester bond or linkages tobe formed, e.g., to a drug or other agent or species. Hydrolysis of theester linkages provides, for example, drug or agent release. It willalso be appreciated by one skilled in the art that the preferred pMAAcan be reacted with many compounds in addition to the DOPA disclosedherein.

Monomers other than tBMA can certainly be used to create the hydrophilicmid-block, whether in a protected or unprotected configuration.Protecting groups such as carbobenzyloxy (Cbz) and tert-butylmethylsilyl(TBDMS) are well known protecting groups for —NH₂ and —OH, respectively.2-methylallylamine and 2-methyllyl alcohol are possible substitutes fortBMA.

One skilled in the art will appreciate that this invention involves thesteps of inducing gel formation by solvent exchange in a multi-blockco-polymer having two or more “blocks.” The copolymer blocks areselected for their hydrophobicity/hydrophilicity to produce gels. Theblocks are also selected, or modified, to incorporate specific andspecified functional groups chosen to control, primarily to enhance,adhesive interactions. Specific embodiments of the invention disclosedherein should not be used narrowly to interpret the more general scopeof this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be illustrated, in its preferredpractice, by the description below. The attached claims should not benarrowly construed in view of the disclosure hereof and of the attachedfigures in which:

FIG. 1 is a chemical structure of synthesized PMMA-PTBMA-PMMA triblockcopolymer obtained by anionic polymerization with sequential monomeraddition using a difunctional initiator;

FIG. 2 is the chemical structure of a converted acrylic triblock polymer(i.e., to PMMA-PMAA-PMMA) such as that shown in FIG. 1;

FIG. 3 is the chemical structure of DOPA modified PMMA-PMAA-PMMA;

FIG. 4 illustrates the assumed molecular structure of the resulting gel;

FIG. 5( a) is a schematic drawing of the sample geometry and (b) is adrawing of the adhesion testing apparatus as used with this invention.The punch radius is a₀ and “h” is the thickness of the elastic layer.

FIG. 6 is a plot of frequency (Hertz, Hz) vs. Young's Modulus(MegaPascal's-MPa) for triblock copolymers of this invention comprising 20%in DMF, NMP, and DMSO, as shown;

FIG. 7 is a plot of polymer fraction vs. Young's Modulus in the samesolvents used in FIG. 6; and

FIG. 8 is a load vs. displacement plot for a hydrogel afterequilibration with a saturated water environment; a hyrdogel of 20%tricopolymer block solution in DMSO.

FIG. 9 load-displacement curves of original and DOPA-modified hydrogelscontracting TiO₂ coated surface in controlled buffer.

FIG. 10 illustrates adhesion of DOPA-modified hydrogels submerged inwater to UV-ozone cleaned (white) or untreated (black) TiO₂ surfaces.The oxidized hydrogels were submerged within a pH 10 solution for fourdays prior to adhesion experiment.

DETAILED DESCRIPTION OF THE INVENTION AND EXAMPLES Triblock CopolymerSynthesis Materials

Methyl methacrylate (MMA) and tert-butyl methacrylate (tBMA) werepurified by addition of triethylaluminum (AlEt₃, Aldrich) solution inhexane until a persistent yellowish color was observed. After degassingby freezing in liquid nitrogen (−78° C.), tBMA was distilled underreduced pressure and stored in freezer whereas MMA was distilleddirectly into the reaction chamber prior to polymerization.Diphenylethylene (DPE, Aldrich) was purified by addition onsec-butyllithium (s-BuLi, Aldrich) until a persistent green color wasobserved. The solution was stirred under nitrogen overnight, anddistilled under reduced atmosphere after degassing, and stored in thefreezer. Difunctional initiator was prepared by the reaction of Li andNaphthalene (both as received) in distilled THF at room temperature for24 hrs under nitrogen atmosphere. As Li reacts with Naphthalene, thecolor of the solution became dark green. LiCl was dried in the reactionchamber at 130° C. under vacuum overnight. Sodium (dispersion inParaffin) and benzophenone were added to the THF, and refluxed until apersistent purple color was observed.

Anionic polymerization of tBMA and MMA (FIG. 1) was carried out under anitrogen atmosphere by using a difunctional initiator. THF was distilledinto the reaction chamber and stirred for 30 minutes to dissolve theLiCl. The concentration of initiator was determined by titration of thegreen initiator solution with a known amount of acetanilide in distilledIHF prior to addition. The chamber was then cooled with a MeOH/dry icebath, and the IHF solution was titrated by adding a few drops ofinitiator until a faint green color was observed. The calculated volumeof initiator was added, and a dark green color was observed immediately.After addition of the DPE, the green color immediately turned into adeep red. The deep red color immediately disappeared when the tBMA wasintroduced dropwise into the reaction flask. The polymerization wasallowed to proceed at −78° C. for 2 hours. A small sample was taken byusing a steel needle just before MMA transfer in order to determine themolecular weight of the tBMA block. MMA was then distilled into thereaction chamber, and the solution was stirred for 1 h beforetermination with anhydrous MeOH. The final solution was concentrated andprecipitated into methanol-water (90:10) mixture under stirring. Thepolymer was dried under vacuum overnight.

The total molecular weight of the polymer as determined by GPC was120,000 g/mole with a polydispersity index of 1.08. The molecular weightof the midblock was 80,000 g/mole. The chemical structure of thistriblock copolymer is shown in FIG. 1.

Conversion of Midblock into Methacrylic Acid

PMMA-PtBMA-PMMA triblock copolymer was completely dissolved in dioxane,and hydrolyzed with hydrochloric acid at 80° C. for 6 hrs. The colorlesssolution became yellowish with time. The solution was precipitated inhexane, and the polymer was washed with hexane and water several timesbefore it was dried under vacuum overnight. After conversion 1 H NMRshowed that the t-C(CH3)3 signal (at 1.43 ppm) had completelydisappeared, indicating that the conversion was complete, giving thepolymer structure shown in FIG. 2.

The PMMA-PMAA-PMMA triblock copolymer synthesized above was completelydissolved in DMF. DOPA methyl ester (DME), 1-hydroxybenzotriazolehydrate (HOBT) and o-benzotriazole-N,N,N_(i) ⁻,N_(i)⁻-tetramethyl-uronium-hexafluoro-phosphate (HBTU) were dissolved in DMFin separate vials and added into the triblock solution in the writtensequential order. The reaction was completed after the addition oftriethylamine (E_(t3)N). All reactions were carried out under nitrogenatmosphere to give the DOPA-containing polymers shown in FIG. 3. Thehydrogel-forming experiments described below were performed on polymersthat do not contain DOPA.

Hydrogel Formation and Characterization Bulk Gel Properties(Characterization)

The triblock copolymer was dissolved in a solvent that is a good solventfor both mid- and end-blocks. The solution was poured into a circularwasher which was attached to a glass slide. Then it was exposed to asaturated water environment for a sufficient period of time to enablewater diffusion into the solution. As water diffuses into the solutionand the original solvent diffuses out, the hydrophobic end-blocksaggregate and form spherical domains. The hydrophilic mid-block formsbridges between these domains, and also loops, as shown schematically inFIG. 4. FIG. 4 is a picture of a hydrogel after equilibration with asaturated water environment (left) and the assumed molecular structureof the gel (right).

Axisymmetric Adhesion Tests

The experimental setup utilized for adhesion experiments anddetermination of the elastic moduls is shown schematically FIG. 5. Aflat punch is driven by an inchworm motor and is attached to a 50 g loadtransducer. A fiber optic displacement sensor is used to measuredisplacement of the punch. FIG. 5 is a schematic drawing of (a) thesample geometry and (b) the adhesion testing apparatus. Note that a, isthe punch radius and b is the thickness of the elastic layer.

The geometry of the mechanical test provides a well defined contactradius that corresponds to the punch radius, a. Young's modulus of thegel, E, is determined from the relationship between the load, P and thedisplacement, δ, utilizing the following expression.

$E = \left. {\frac{3P}{8a\; \delta}\left\lbrack {1 + {1.33\left( \frac{a}{h} \right)} + {1.33\left( \frac{a}{h} \right)^{3}}} \right\rbrack}^{- 1} \right|$

The energy release rate (G) can be calculated from the followingequation:

$G = \frac{3\left( P_{t} \right)^{2}}{32\pi \; E\; a^{3}}$

where P_(t) is the measured tensile load.

The frequency-dependent dynamic moduli are measured by applying asinusoidally varying displacement to the sample.

Measured values of the elastic modulus are plotted in FIG. 6. Gels wereprepared as described before, and exposed to humidity in a closedenvironment until the size and elastic properties of the gels no longerchanged with time (typically 3 days). The moduli range from 10 to 30MPa, depending on the initial solvent that was used. FIG. 6 shows themagnitude of the complex Young's modulus at different frequencies forhydrogels that were formed from the triblock formed in 20% solution ofDMF, NMP and DMSO. Equilibrated gel thicknesses were around 2 mm.

The relation between elastic modulus and equilibrium polymerconcentrations of the hydrogels is given in FIG. 7. It is clear thatelastic modulus increases with increasing polymer concentration, andthat the final polymer concentration depends on the solvent in which thetriblock copolymer was originally dissolved. FIG. 7 shows Young'sModulus at 0.1 Hz plotted against final polymer concentration afterequilibration with saturated water vapor.

Preliminary adhesion experiments were also performed by using a steelpunch having a radius of 0.39 mm. A load-displacement plot for a sampleprepared from DMSO is shown in FIG. 8. The punch was brought intocontact with the gel, and a maximum compressive load of 5 mN was appliedfor 5 minutes. A tensile force was observed when the punch was thenretracted from the gel, indicating that there is some adhesion betweenthe steel punch and the hydrogel, even in the absence of DOPA.

FIG. 8 is a load-displacement curve for hydrogel after equilibrationwith a saturated water environment. The hydrogel was formed from a 20%solution of the triblock copolymer in DMSO.

PMMA-PMAA-PMMA triblock copolymers can be completely dissolved intoxicologically acceptable solvents such as NMP, EtOH and DMSO, as wellas other solvents such as MeOH and DMF. Hydrogels are formed by a simplesolvent exchange mechanism, during exposure of polymer solutions towater vapor. The elastic moduli are relatively high (^(˜)15-30 MPa),which is consistent with the relatively high polymer volume fractions inthe gel after the solvent exchange process is completed.

DOPA-modified hydrogels were prepared in the same manner from DMSOsolutions of DOPA-modified copolymer. The gel obtained after solventexchange was de-swollen and opaque, and did not swell at all whenimmersed in neutral water. This behavior is attributed to the relativelyhydrophobic character of the DOPA moieties in the midblock. In order toobtain swollen gels, they were immersed in pH10 buffer solutions aftersolvent exchange. The gel swelled, became a transparent red, and thenbecame a deep red, which is an indication of DOPA oxidation. The modulusof the swollen DOPA-hydrogel was found to be 1.3 kPa by indentationmethod.

Axisymmetric Adhesion Tests

Adhesion of DOPA-modified hydrogel in contact with TiO₂ was measuredwith the indentation method. A flat punch coated with TiO₂ was broughtinto contact with the hydrogel, and a maximum compressive load of 5 mNwas applied. The load was retracted until the surfaces were separated.Contact curves of original hydrogel (without DOPA) show very littlehysteresis, as shown in FIG. 5. On the other hand, a significantnegative load (tensile load, Pt) was developed when the TiO2-coatedpunch was retracted from the gel, an indication of adhesion betweenhydrogel surface and the metal oxide surface. For the DOPA modifiedhydrogel we calculate a critical energy release rate, Gc of≈27 mJ/m2,from the maximum tensile load at pulloff. Young's modulus (E) of thegels was obtained from the slope of advancing portion of loaddisplacement data, according to Eq. 2. Results are summarized in Table 1(below).

In this study a polymer system was developed that was intended to mimicmussel adhesive proteins, and was capable of self assembling into anadhesive hydrogel when injected into an aqueous environment. DOPAmodified PMMA-PMAA-PMMA triblock copolymers described in this study arepotential candidates for in situ gel forming bioadhesive materialssuitable for tissue repair and regeneration.

Table 1 properties of gels initially prepared from DMSO solution, andequilibrated in controlled buffer (pH=10). Φin and Φp are the respectivepolymer concentrations of the initial solution prior to solventexchange, and the swollen gel equilibrated in buffer solution.

Hydrogel Φin Φp E (kPa) G (mJ/m2) Original 0.1 0.01 1.6 — DOPA Modified0.2 0.05 2.6 27

Highly swollen hydrogels were formed for pH values greater than 4, withG≈1 kPa. This is attributed to ionization of the methacrylic acidmid-blocks. At neutral pH the gels formed from the triblock with highDOPA content was stiffer, and opaque. A transparent red gel was obtainedby immersing the preformed gel in pH=10 buffer. The red color is anindication of DOPA oxidation. These DOPA modified hydrogels had amodulus of 2.6 kPa. The presence of oxidized DOPA significantlyincreased the adhesion to TiO2 surfaces that had been immersed in water.

FIG. 10 shows the amount of energy required to separate theDOPA-modified hydrogels from wetted TiO₂ surfaces. The adhesion energyincreased with increasing DOPA content in the block copolymer on bothUV-ozone treated and untreated TiO₂ surfaces. This experiment alsodemonstrated that the unoxidized form of DOPA is responsible for thewater-resistant adhesion as the adhesive strength of the oxidizedhydrogels was reduced by over 20 fold.

REFERENCES INCORPORATED HEREIN BY REFERENCE

The following publications are incorporated by reference herein:

“Synthesis and Adhesion Properties of DOPA Incorporated Acrylic TriblockHydrogels”, Guvendiren, Murat, Bruce P. Lee, Phillip B. Messersmith andKenneth R. Shull, Department of Materials Science and Engineering,Northwestern University, Evanston, Ill.; Department of BiomedicalEngineering, Northwestern University, Evanston, Ill.

Tae G., Kornfield J. A., Hubbel J. A., Biomaterials, 2005, 26,5259-5266.

Yu M., Hwang J., and Deming T. J., J.Am Chem. Soc, 1999, 121, 5825-5826.

Shull K. R., Mat.Sci.Eng., 2002, R36, 1-45. Webber R. E., et al.,Physical Review E, 2003, 68, 021805. Crosby A. J., et al., J. Rheology,2002, 46, 273.

“Alpha, Beta-Dehydro-3,4-Dihydroxyphenylalanine Derivatives—PotentialSchlerotization Intermediates in Natural Composite—Materials”, Rzepecki,L. M., Nagafuchi, T., and Waite, J. H., Arch. Biochem. Biophys. 1991,285, 17-26.

“Hydroxyarginine-Containing Polyphenolic Proteins in the AdhesivePlaques of the Marine Mussel Mytilus-Edulis”, Papov, V. V., Diamond, T.V., Biemann, K., and Waite, J. H., J BioL Chem. 1995, 270, 20183-92.

“Wresting the muscle from mussel beards: Research and applications”,Rzepecki, L. M. and Waite, J. H., Mol. Mar. Biol. Biotechnol. 1995, 4,313-22.

Waite, J. H., in Redox-Active Amino Acids in Biology, 1995, Vol. 258, p.1-20.

“Enzymatic tempering of a mussel adhesive protein film”, Hansen, D. C.,Corcoran, S. G., and Waite, J. H., Langmuir 1998, 14, 1139-47.“Mytilus edulis adhesive protein (MAP) as an enzyme immobilizationmatrix in the fabrication of enzyme-based electrodes”, Saby, C. andLuong, J. H. T., Electroanalysis 1998, 10, 1193-9.“Synthetic polypeptide mimics of marine adhesives”, Yu, M. E. andDeming, T. J., Macromolecules 1998, 31, 4739-45.“Mussel byssus and biomolecular materials”, Denning, T. J., CurrentOpinion In Chemical Biology 1999, 3, 100-5.“Expression of multiple forms of an adhesive plaque protein in anindividual mussel, Mytilus edulis”, Warner, S. C. and Waite, J. H., Mar.Biol. 1999, 134, 729-34.“Synthesis and characterization of self-assembling block copolymerscontaining adhesive moieties”, Huang, K., Lee, B., and Messersmith, P.B., Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 2001, 42, 147-8.“Enzymatic and non-enzymatic pathways to formation of DOPA-modified PEGhydrogels”, Lee, B. P., Dalsin, J. L., and Messersmith, P. B., Polym.Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 2001, 42, 151-2.“Synthesis of 3,4-dihydroxyphenylalanine (DOPA) containing monomers andtheir co-polymerization with PEG-diacrylate to form hydrogels”, Lee, B.P., Huang, K., Nunalee, N., Shull, K. R., and Messersmith, P. B., JBiomater. Sci. Polymer Ed. 2004, 15, 449-64.

E. Ruel-Gariepy and J.-C. Leroux, European Journal of Pharmaceutics andBiopharmaceutics, 2004, 58, 409-426. S. Dai, P. Ravi, K. C. Tam, B. W.Mao and L. H. Gan, Langmuir, 2003, 19, 5175-5177. A. Rozier, C. Mazuel,J. Grove and B. Plazonnet, International Journal of Pharmaceutics, 1989,57, 163-168. G. Tae, J. A. Kornfield and J. A. Hubbell, Biomaterials,2005, 26, 5259-5266. B. P. Lee, C.-Y. Chao, F. N. Nunalee, E. Motan, K.R. Shull and P. B. Messersmith, Macromolecules, 2006, 39, 1740-1748.

B. P. Lee, J. L. Dalsin and P. B. Messersmith, Biomacromolecules, 2002,3, 1038-1047.

1. An anionically polymerized block copolymer comprising the formula: